The ideal tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, will be a highly effective vaccine that prevents blood stage infection and thereby prevents all disease and transmission. Sanaria's goal is to develop and commercialize a Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in > 90% of recipients. Immunization of humans by the bites of > 103 mosquitoes introducing attenuated PfSPZ can elicit > 90% protection against infection challenge. Based on these data, Sanaria developed a vaccine composed of attenuated, purified, aseptic, cryopreserved PfSPZ, the PfSPZ Vaccine. In the 1st Phase 1 clinical trial the PfSPZ Vaccine was administered to 80 volunteers subcutaneously (SC) or intradermally (ID) and shown to be safe, well tolerated, and moderately immunogenic. However, protection against infection was low, raising questions about immunogen potency and route of administration. Subsequent studies in non-human primates (NHPs) and mice have unequivocally demonstrated that the SPZ in the PfSPZ Vaccine are highly potent, and that the poor immunogenicity was due to inefficient administration of PfSPZ. In the next clinical trials at NIH and in Tanzania, the PfSPZ Vaccine will be administered IV. In the Phase II SBIR and supplement that lead to this Competitive Renewal, we aseptically produced and cryopreserved purified PfSPZ from 3 different Pf strains, and manufactured under cGMPs a lot of one of these strains, designated PfSPZ Challenge, for infection of volunteers by ID inoculation. This work was done to facilitate the testing of malaria vaccines against challenge with multiple strains of Pf, and to create the parasite components of a multi-strain PfSPZ vaccine, if necessary. In this Competitive Renewal: lots of PfSPZ Challenge from the 2 additional strains; determine if each of the 3 strains is equally immunogenic and antigenic for CD8+ T cell induction in NHPs, and whether one of the strains is more broadly immunogenic against all strains. We also begin the development of a new approach to vaccine development called PfSPZ Challenge Vaccine, in which individuals undergoing malaria chemoprophylaxis are inoculated with PfSPZ (i.e. PfSPZ Challenge) as the approach to immunization. Each of the following capabilities - 1) to challenge volunteers immunized with PfSPZ Vaccine with multiple strains of PfSPZ Challenge by ID inoculation in multiple clinical trials centers; 2) to determine whether the immunogenicity and antigenicity of PfSPZ (PfNF54) is broadly reactive, and if not, whether Pf7G8 or PfNF135.C10 is more broadly reactive; and 3) to initiate PfSPZ Challenge Vaccine development - has the potential to reduce the time to market for the attenuated PfSPZ Vaccine or the non- attenuated PfSPZ Challenge Vaccine by at least 2 years, thereby reducing development costs by tens to perhaps hundreds of millions of dollars. PUBLIC HEALTH RELEVANCE: To protect individuals against malaria and to aid in eradicating this disease, a highly effective vaccine is needed. Sanaria is in the process of developing such a vaccine: a live attenuated whole organism vaccine called the Plasmodium falciparum sporozoite (PfSPZ) Vaccine. This proposal aims to broaden the protective efficacy of the PfSPZ Vaccine by incorporating multiple strains of Pf SPZ derived from different geographic areas, and to speed progress towards licensure. The results of this study will help to make the vaccine universally effective and available more quickly for travelers and African children.